Project Summary The non-pregnant uterus generates peristalsis in a menstrual cycle dependent manner under the control of sex hormones. This motility is a driving force to move menses, transport sperm and embryo, and regulate implantation; hyper- or dysfunctional peristalsis has been linked to major gynecological and obstetric disorders such as endometriosis and adenomyosis. However, the molecular mechanisms driving uterine peristalsis remain unclear. With state-of-the-art two-photon imaging of Ca2+ signals in a novel precision-cut uterine slice preparation, we found that myometrial cells generate highly active asynchronous Ca2+ oscillations (ACaOs) and synchronous Ca2+ oscillations (SCaOs) in an extracellular Ca2+ dependent manner. At the whole slice level, we further found that they generate rhythmic contraction consisting of a phasic component added to a basal tone, each dependent on extracellular Ca2+. Moreover, a T-type channel blocker inhibits ACaOs but not ScaOs; an L-type channel blocker and a TMEM16A (Cl- channel) antagonist inhibit SCaOs but not ACaOs. In light of the isoform expression levels and biophysics of the aforementioned three channels in uterus, we hypothesize that the Cav3.2 T type channel is responsible for ACaOs and basal tone, and the interplay of Cav1.2 and TMEM16A generates SCaOs and phasic contraction. To test and establish these hypotheses, we will fully characterize ACaOs and SCaOs with two-photon imaging of uterine slices. Mice with smooth muscle specific deletion of TMEM16A or Cav1.2 and systemic deletion of Cav3.2 will be used to firmly establish the role each channel plays in these Ca2+ events and in force generation. This study will establish the ACaOs and SCaOs as two key signals responsible for uterine peristalsis and identify the gene(s) for each component. The outcome of this study will establish the basis to target genes in myometrial cells for new treatments for reproduction disorders such as endometriosis and adenomyosis.